1 / 35

Laboratory of Physiology KULeuven Leuven, Belgium

REGULATION OF INTRACELLULAR Ca 2+ RELEASE BY CALMODULIN AND CALMODULIN-LIKE Ca 2+ -SENSOR PROTEINS. Laboratory of Physiology KULeuven Leuven, Belgium. Localization of IP 3 R1 and IP 3 R3 in A7r5 cells. IP 3 R1. IP 3 R3. Cytoplasm. Perinuclear region. Agonists

lilly
Download Presentation

Laboratory of Physiology KULeuven Leuven, Belgium

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. REGULATION OF INTRACELLULAR Ca2+ RELEASE BY CALMODULIN AND CALMODULIN-LIKE Ca2+ -SENSOR PROTEINS Laboratory of Physiology KULeuven Leuven, Belgium

  2. Localization of IP3R1 and IP3R3 in A7r5 cells IP3R1 IP3R3 Cytoplasm Perinuclear region

  3. Agonists IP3 Ca2+ Plasma membrane associated: Homer-mGluR TRPs; RhoA-TRPC1 G Cytoskeletal proteins: Actin; MyosinII Ankyrin; Tallin; Vinculin 4.1N Kinases and phosphatases: PKA; Fyn BANK- PTK IRAG-PKG FKBP12-Calcineurin PP1 Cytosolic proteins: Calmodulin; CaBP IRBIT; CARP Intraluminal proteins: Chromogranins; Calnexin IP3R I, II, III

  4. IICR is dependent on luminal and cytosolic Ca2+

  5. Effect of Calmodulin on IP3-induced Ca2+ release A7r5 cells 70% IP3R1 Control CaM Control CaM HBE cells 90% IP3R3 A7r5 HBE

  6. IP3-binding domain Coupling domain Channel domain CaM SPA technique: Adkins et al., 2000 Ca2+

  7. Lbs-1: IP3 binding core (226-604) aa 1-225 = suppressor NH2 CaM CYT Ca2+CaM COOH ER Recombinant ligand-binding domain of IP3R1 (LBS-1) 1 581 Lbs-1 1-225 W226 581 Lbs-1 1-225

  8. 581 1 Lbs-1His W226 581 Lbs-1 1-225His EC50= 1.7µM [3H]IP3 binding (%) 0.3 B/F 0.2 0.1 control 5 µM Ca2+ Ca2+ CaM Ca2+ CaM1234 10 µM apoCaM 10 µM CaM1234 0.0 0 5 10 Bound (nM)

  9. 1 581 Lbs-1 W226 581 Lbs-1 1-225 309 159 1 Cyt1 Cyt2 50 M free Ca2+ 1 mM EGTA Localisation of the N-terminal Calmodulin-Binding Site GST-fusion protein pull down of CaM1234 GST GST-Cyt1 GST-Cyt2

  10. 159 1 1-5-10 1-5-10 53% IQ 1-5-8-14 76% IQ 70% IQ (site1) A C D E B F A B C D E F CaM A B C D E F CaM 1,0 200 µM free Ca2+ 1 mM EGTA 0,8 1- B/Bo 0,6 0,4 0,2 0,0 -0,2 A B C D E F CaM CaM-binding in the N-terminal region EGTA Ca2+ Ca2+ independent EC50 1-1.5 µM

  11. 1499 1649 76 % IQ 1-5-8-14 72% 1-5-8-14 60 % IQ G ldsqvnnlflkshnivqkta ldsqvnnlflkshnivqktalmwrlsarnaar kshnivqktalmwrlsarnaar kshnivqktalmwrlsarnaarrdsvlaasrd H I J 50 µM Ca2+ 1 mM EGTA 2.0 1.5 1.0 Peptides CaM-binding in the coupling region F/F0 Ca2+ dependent EC50 60 nM Ca2+ independent EC50 200 nM G H I J

  12. Calmodulin binding sites on IP3R1 Cytosol R1:PPKKFRDCLFKLCPMNRYSAQKQFWKAAKPGAN R2:PPKKFRDCLFKVCPMNRYSAQKQYWKAKQAKQG R3:PPKKFRDCLFKVCPMNRYSAQKQYWKAKQTKQD CaM W1577A (Zhang et al, 2001; Nosyreva et al, 2002) R1:LDSQVNNLFLKSHN-IVQKTAMNWRLSARN-AARRDSVLA R2:LDSQVNTLFMKNHSSTVQRAAMGWRLSARSGPRFKEALGG R3:LDAHMSALLSSGGSCSAAAQRSAANYKTATRTFPRVIPTA 31 25 13 18 Endoplasmic reticulum Ca2+/CaM

  13. CaM CaM 1234 Control CaM IICR is inhibited by CaM as well as by CaM1234 0.5 1 100 ATP (µM ) 600 Control 400 Ca2+i (nM) 200 0 0 250 500 750 1000

  14. Inhibitory Activatory ?(Yang et al., 2002) Adapted from Haeseleer et al., 2000 CaM or other CaM-like Ca2+ sensor proteins ?

  15. aa 1-225 (suppressor) IP3 binding core (226-604) NH2 CaM GST GST 1-604 GST 1-225 GST 226-604 CYT CaBP COOH ER CaBP binds to the InsP3R

  16. CaBP binds to a similar region of the InsP3R as CaM A) 159 CaM CaM 1 A C D E B F B) + Ca2+ -Ca2+/ EGTA sCaBP1 C D F A B E C D F A B E sCaBP1

  17. 1.0 Band intensity 0.5 0.0 0 2 4 6 8 10 Peptide B: CaBP Binding of CaBP to the InsP3R is calcium independent Ratio of CaBP: peptide B CaBP CaBP 1/10 1/1 1/2 1/3 1/4 1/5 1/6 1/8 + Calcium + EGTA

  18. CaBP inhibits InsP3 binding to the InsP3R 4 µg Lbs-1 10 µM sCaBP1 5 µM Ca2+ 100% 100 78 ± 1.9 % 68 ± 6.9 % 75 [3H]IP3 Binding (% vs control) 39 ± 4.7% 50 25 0 sCaBP1 Ca Ca/ sCaBP1 Lbs-1

  19. SCaBP LCaBP Both Long and Short CaBPs inhibit InsP3 induced Calcium release 100µM 1µM 0.5µM ATP 1000 800 600 Ca2+i (nM) 400 Control 200 0 0 200 400 600 800 1000 Time (s)

  20. EF1 EF2 EF3 EF4 0.5 1 100 µM ATP 100 1000 800 % responsive cells 50 Control 600 CaBP134 Ca2+i (nM) 400 0 0.5 1 100 200 ATP (mM) 0 0 1500 500 1000 Time (s) CaBP inhibits InsP3 induced Calcium release independent of Calcium binding

  21. TRP CIRB sCaBP-1 GST / CaM AdPhos IP3 1-->225 Lbs-1: IP3 binding core (IBC 226-604) aa 1-225 CaM NH2 CYT Ca2+CaM COOH Interaction 1-225 with IBC ER

  22. IP3R1 + Suramin + Suramin CaM-Seph CaM-Seph Seph Ca 2+ Seph EGTA Input Control 1-225 10 µM CaM Ca 2+ EGTA Suramin Interacts with the CaM-binding sites on the IP3R1 Control 100 µM suramin

  23. Suramin induces a large IP3-independent Ca2+ release in A7r5 cells

  24. New type of Ca2+ -activated Ca2+ release channel ?? + CaM1234

  25. Ca2+ dependence: EC50 = 700 nM Hill = 1.9 Mg2+ inhibition: EC50= 0.6 mM ATP stimulation: EC50= 320 µM Characteristics of the CICR mode

  26. Effects of CaM, CaM1 and CaM1234 40 30 20 Fractional loss (%/ 2 min) control 10 0 0 10 20 Time (min) control CaM1 CaM CaM1234

  27. Calmodulin Calmodulin1 Calmodulin1234 Long CaBP1 Short CaBP1 Short CaBP1 NCS NCS NCS NCS - - - - 1/Frequenin 1/Frequenin 1/Frequenin 1/Frequenin NCS NCS - - 1/FrequeninE120Q 1/FrequeninE120Q Calmodulin Calmodulin Calmodulin1 Calmodulin1234 Calmodulin1234 Long CaBP1 Long CaBP1 Short CaBP1 NCS - 1/FrequeninE120Q

  28. Preincubation with a CaM-binding peptide inhibits CICR control Ca2+ (3 µM)

  29. Preincubation with a CaM-binding peptide inhibits CICR control RyR CaM-BS (peptide aa 3614-3643) Ca2+ (3 µM)

  30. CaM but not CaM1234 can restore CICR Preincubation with RyR CaM-BS (peptide aa 3614-3643) Ca2+ (3 µM)

  31. CaM but not CaM1234 can restore CICR CaM1234 Preincubation with RyR CaM-BS (peptide aa 3614-3643) CaM Ca2+ (3 µM)

  32. IP3Rs a subgroup of cellular Ca2+ channels IP3R1IP3R2IP3R3 RYR1RYR2RYR3 Other types? Other types?

  33. New type of Ca2+ -activated Ca2+ release channel ?? CaM1234 Mg2+ - - Ca2+ ATP + + CaM IP3R CICR Cahalan MD, Nature Cell Biology, 2002 Defect gene in polycystic kidney disease is a CICR channel located in the intracellular membrane Ancestral type of CICR channelrelated to polycystin-2 ??inhibited by apoCaM and CaM-like

  34. KULeuven Leuven, Belgium Babraham Institute Cambridge UK Jan B. PARYS Geert CALLEWAERT Ludwig MISSIAEN Rafael A. FISSORE Nael NADIF KASRI Geert BULTYNCK Karolina SZLUFCIK Kristel VAN ACKER Leen VERBERT Elke VERMASSEN Zerihun ASSEFA Patrick DE SMET H. Llewelyn Roderick Martin D. Bootman Michael Berridge

More Related